Neurosteroids and enantiomers thereof for the prevention and treatment of neurodegenerative conditions

ABSTRACT

The present disclosure is directed to a composition for prevention or treatment of neurodegenerative conditions. The composition comprises a neurosteroid, a synthetic enantiomer of a neurosteroid, or a combination thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/782,406, filed on Feb. 5, 2019, which claims priority to U.S.Provisional Application No. 62/801,187, filed Feb. 5, 2019, the contentsof which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under MH101874 andMH077791 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

Glaucoma is a leading cause of irreversible blindness, and involvesselective damage to retinal ganglion cells (RGCs). Increased intraocularpressure (IOP) is a major risk factor for glaucoma. However, thepathogenesis underlying RGC damage by IOP elevation remains unclear.

Autophagy is an intracellular degradation system induced under cellularstress to digest cytoplasmic constituents or cell organelles to maintainnutrient and energy homeostasis. Autophagy begins with an expandingdouble-membrane structure called a phagophore or isolation membrane inthe cytoplasm. The isolation membrane sequestering cytosolic materialsand organelles forms a double-membrane vesicle called an autophagosome(AP). APs fuse with the lysosome to degrade their contents, then becomedegenerative autophagic vacuoles (DAVs) (FIG. 1A). Autophagy alsomaintains intracellular homeostasis by eliminating damaged cellorganelles and misfolded proteins, and plays an important role inneurodegenerative diseases including glaucoma, age-related maculardegeneration, retinal vascular occlusion, diabetic retinopathy,Alzheimer's disease and traumatic brain injury.

In general, a neurosteroid is an endogenous or non-endogenous steroid orsteroid analogue with the absolute configuration of natural steroids ortheir unnatural mirror images (enantiomers) that modulate central orperipheral nervous system function. Enantiomers of natural steroids donot occur in nature, accordingly steroid enantiomers are synthetic.Endogenous neurosteroids are modulators generated in the nervous systemin response to cellular stress and are potent modulators ofneurotransmitter systems. For purposes of this disclosure, the term“neurosteroid” is used to designate any neurosteroid, any neuroactivesteroid, any steroid that is made locally in the brain, any steroid notmade in the brain but made elsewhere in the body that alters brainfunction, and any synthetic steroid that alters brain function.Neurosteroids and enantiomeric neurosteroids include, but are notlimited to allopregnanolone, cholesterol, pregnenolone, progesterone,pregn-5α-ane-3,17-dione, pregn-5β-ane-3,17-dione, androsterone,etiocholanolone, tetrahydrodeoxycorticosterone, and any of theneurosteroids disclosed in U.S. Pat. No. 10,202,413 B2, which is hereinincorporated by reference.

Among them, allopregnanolone (AlloP) is a strong enhancer of GABA_(A)receptors. AlloP attenuates pressure-induced retinal injury in ex vivorat retinas. Because neuroprotective effects of AlloP were inhibited bya specific GABA_(A) antagonist, the neuroprotection with AlloP is likelymediated by GABAergic signaling. However, neuroprotection by AlloP maynot exclusively involve GABA_(A) receptors. AlloP was found to activateautophagy in a mouse model of Niemann-Pick Type C disease and in primaryastrocyte cultures, suggesting that upregulation of autophagic flux maycontribute to endogenous neuroprotective mechanisms. Ent-AlloP is thesynthetic enantiomer of AlloP (FIG. 1B) and has weak actions on GABA_(A)receptor signaling. In spite of its differences from AlloP, ent-AlloPmay have neuroprotective effects in a mouse model of Niemann-Pick Type Cdisease, raising the possibility that ent-AlloP acts via mechanismsdistinct from AlloP.

As described herein, a rat ex vivo ocular hypertension (OHT) model witha closed chamber incubation system (FIG. 1C) and an in vivo OHT modelfollowing injection of polystyrene microbeads into the anterior chamberwere used to compare neuroprotective effects of AlloP and ent-AlloP,focusing on the role of GABA_(A) receptors and autophagy.

BRIEF DESCRIPTION OF THE DISCLOSURE

In one aspect, the present disclosure is directed to a composition forprevention or treatment of a neurodegenerative condition. Thecomposition comprises a neurosteroid, a synthetic enantiomer of aneurosteroid, or a combination thereof.

In another aspect, the present disclosure is directed to a method ofpreventing or treating a neurodegenerative condition comprising inducingautophagy. The method comprises administering an effective amount of acomposition comprising a neurosteroid, a synthetic enantiomer of aneurosteroid, or a combination thereof.

In yet another, aspect, the present disclosure is directed to a methodof attenuating pressure-induced retinal injury. The method comprisesadministering an effective amount of a composition comprising aneurosteroid, a synthetic enantiomer of a neurosteroid, or a combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments described herein may be better understood by referringto the following description in conjunction with the accompanyingdrawings.

FIGS. 1 (A-S) are exemplary embodiments of autophagy flux andneuroprotective effects of neurosteroids on pressure-mediated retinaldegeneration in an ex vivo model in accordance with the presentdisclosure. FIG. 1A discloses key steps of autophagy flux. FIG. 1Bdiscloses structures of neurosteroids. FIG. 1C discloses a closedpressure-loading system. FIG. 1D discloses a light micrograph ofpressure-loaded retina at 10 mmHg (arrow indicates blood capillary).FIG. 1E discloses a light micrograph of pressure-loaded retina at 75mmHg (arrowheads indicate axonal swelling). FIG. 1F discloses a lightmicrograph of pressure-loaded retina at 75 mmHg with 1 μM AlloP. FIG. 1Gdiscloses a light micrograph of pressure-loaded retina at 75 mmHg with 1μM ent-AlloP. FIG. 1H discloses a light micrograph of pressure-loadedretina with administration of 1 μM AlloP and 1 μM picrotoxin. FIG. 1Idiscloses a light micrograph of pressure-loaded retina withadministration of 1 μM ent-AlloP and 1 μM picrotoxin, scale bars 20 mm.FIG. 1J discloses RGC survival and neuroprotection in pressure-loadedwhole mounted retinas at 10 mmHg. FIG. 1K discloses RGC survival andneuroprotection in pressure-loaded whole mounted retinas at 75 mmHg.FIG. 1L discloses RGC survival and neuroprotection in pressure-loadedwhole mounted retinas with 1 μM AlloP at 75 mmHg. FIG. 1M discloses RGCsurvival and neuroprotection in pressure-loaded whole mounted retinaswith 1 μM ent-AlloP at 75 mmHg, scale bars 200 FIG. IN discloses thenumber of NeuN-positive cells in whole mount retinas (Tukey *p<0.05).FIG. 1O discloses 10 mmHg TUNEL staining. FIG. 1P discloses 75 mmHgTUNEL staining. FIG. 1Q discloses TUNEL staining with 1 μM AlloP. FIG.1R discloses TUNEL staining with 1 μM ent-AlloP, scale bars 30 FIG. 1Sdiscloses the number of TUNEL-positive RGCs per 200 μm of retina section(Tukey *p<0.05).

FIGS. 2 (A-S) are exemplary embodiments of electron micrographic andWestern blot analyses of ex vivo retinas in accordance with the presentdisclosure. FIG. 2A discloses an electron micrographic of an ex vivoretina at 10 mmHg (“Mu” indicates Muller cell). FIG. 2B discloses anelectron micrographic of an ex vivo retina at 75 mmHg (arrowheadsindicate swollen axons). FIG. 2C discloses an electron micrographic ofan ex vivo retina with AlloP (1 μM) at 75 mmHg (“NFL” indicates nervefiber layer). FIG. 2D discloses an electron micrographic of an ex vivoretina with ent-AlloP (1 μM) at 75 mmHg. FIG. 2E discloses a DAV(indicated by double arrows) in the NFL at 10 mmHg. FIG. 2F discloses APin the NFL at 10 mmHg. FIG. 2G discloses a DAV in the NFL at 10 mmHg.FIG. 2H discloses a DAV (indicated by double arrows) in the NFL at 75mmHg. FIG. 2I discloses a DAV in the NFL at 75 mmHg. FIG. 2J disclosesan isolation membrane in the NFL at 75 mmHg. FIG. 2K discloses 1 μMAlloP with AP indicated by single arrow. FIG. 2L discloses 1 μM AlloPwith DAV indicated by double arrows. FIG. 2M discloses 1 μM ent-AlloP at75 mmHg with DAV indicated by double arrows. FIG. 2N discloses thenumber of AP per 50 μm2 of retina. (Dunnett

p<0.05 or Tukey *p<0.05). FIG. 2O discloses the number of DAV per 50 μm2of retina (Dunnett

p<0.05 or Tukey *p<0.05). FIG. 2P discloses representative Western blotanalyses of LC3. FIG. 2Q discloses quantitative Western blot analysis ofLC3II expression (Dunnett

p<0.05 or Tukey *p<0.05). FIG. 2R discloses representative Western blotanalyses of p62. *p<0.05. FIG. 2S discloses quantitative Western blotanalysis of p62 expression (Dunnett

p<0.05 or Tukey *p<0.05).

FIGS. 3 (A-H) are exemplary embodiments of effects of pressureelevation, neurosteroids, and 3-MA in accordance with the presentdisclosure. FIG. 3A discloses a light micrograph of retinal morphologyfor administration of 10 mM 3-MA with no differences in the retinaincubated with 1 μM AlloP at 75 mmHg. FIG. 3B discloses a lightmicrograph of retinal morphology for administration of 10 mM 3-MA withdampened protective effects of 1 μM ent-AlloP at 75 mmHg (arrowheadsindicate axonal swelling; arrows indicate RGC degeneration). FIG. 3Cdiscloses a light micrograph of retinal morphology with severe induceddamage by 3-MA (10 mM) at 75 mmHg. FIG. 3D discloses a light micrographof retinal morphology with induced degeneration by 3-MA (10 mM) at 10mmHg, scale bars, 20 FIG. 3E discloses representative Western blotanalyses of LC3 proteins in pressure-loaded retinas (75 mmHg) treatedwith 1 μM ent-AlloP alone or with 1 μM ent-AlloP and 10 mM 3-MA. FIG. 3Fdiscloses quantitative Western blot analysis of LC3-II expression in theretina incubated with ent-AlloP at 75 mmHg (Wilcoxon Rank-Sum Test*p<0.05). FIG. 3G discloses representative Western blot analyses of p62proteins in pressure-loaded retinas (75 mmHg) treated with 1 μMent-AlloP alone or with 1 μM ent-AlloP and 10 mM 3-MA. FIG. 3H disclosesquantitative Western blot analysis of p62 expression.

FIGS. 4 (A-S) are exemplary embodiments of effects of IOP andneurosteroids on RGC or axonal survival in accordance with the presentdisclosure. FIG. 4A discloses an injection of microbeads (surroundedwith a broken line) into the anterior chamber. FIG. 4B discloses a lightmicrograph of the iridocorneal angle (“TM” indicates trabecularmeshwork; “CB” indicates ciliary body). FIG. 4C discloses an enlargementof the rectangular area in FIG. 4B. FIG. 4D discloses IOP profiles. FIG.4E discloses control eye TUNEL staining. FIG. 4F discloses non-treatedOHT eye TUNEL staining. FIG. 4G discloses TUNEL staining foradministration of 1 μM AlloP in OHT eyes, scale bars 30 FIG. 4Hdiscloses TUNEL staining for administration of 1 μM ent-AlloP in OHTeyes, scale bars 30 FIG. 4I discloses the number of TUNEL-positive RGCsper 200 μm of retinal sections (Tukey *p<0.05). FIG. 4J discloses aconfocal image of NeuNlabeled RGCs in a Control eye. FIG. 4K discloses aconfocal image of NeuNlabeled RGCs in a microbead-injected OHT eyewithout neurosteroid administration. FIG. 4L discloses a confocal imageof NeuNlabeled RGCs with administration of 1 μM AlloP or FIG. 4Mdiscloses a confocal image of NeuNlabeled RGCs with administration of 1μM ent-AlloP, scale bars 200 FIG. 4N discloses the number ofNeuN-positive cells in the whole mount retina under each condition(Tukey *p<0.05). FIG. 4O discloses a light micrograph of the optic nerveaxons three weeks after microbead injection for a control eye. FIG. 4Pdiscloses a light micrograph of the optic nerve axons three weeks aftermicrobead injection for microbead-injected eyes with OHT. FIG. 4Qdiscloses a light micrograph of the optic nerve axons three weeks aftermicrobead injection with administration of 1 μM AlloP. FIG. 4R disclosesa light micrograph of the optic nerve axons three weeks after microbeadinjection with administration of 1 μM ent-AlloP, scale bars 10 μm. FIG.4S discloses axonal number in whole mount retinas under each condition(Tukey *p<0.05).

FIGS. 5 (A-Z) are exemplary embodiments of electron micrographs in invivo OHT eyes in accordance with the present disclosure. FIG. 5Adiscloses an electron micrograph of the NFL of a control eye at lowmagnification. FIG. 5B discloses an electron micrograph of DAV of thecontrol eye of FIG. 5A. FIG. 5C discloses an electron micrograph of theisolation membrane of the control eye of FIG. 5A. FIG. 5D discloses anelectron micrograph of the NFL of a non-treated OHT eye at lowmagnification. FIG. 5E discloses an electron micrograph of DAV of thenon-treated OHT eye of FIG. 5D. FIG. 5F discloses an electron micrographof AP of the non-treated OHT eye of FIG. 5D. FIG. 5G discloses anelectron micrograph of the NFL of an OHT eye treated with AlloP at lowmagnification. FIG. 5H discloses an electron micrograph of DAV of the ofOHT eye treated with AlloP of FIG. 5G. FIG. 5I discloses an electronmicrograph of AP (indicated by arrowhead) and DAV (indicated by doublearrows) of the OHT eye treated with AlloP of FIG. 5G. FIG. 5J disclosesan electron micrograph of the NFL of an OHT eye treated with ent-AlloPat low magnification. FIG. 5K discloses an electron micrograph of DAV ofthe OHT eye treated with ent-AlloP of FIG. 5J. FIG. 5L discloses anelectron micrograph of DAV of the OHT eye treated with ent-AlloP of FIG.5J.

FIG. 5M discloses the number of APs (n=4 per experiment, Dunnet †p<0.05,Tukey *p<0.05). FIG. 5N discloses the number of DAVs (n=4 perexperiment, Dunnet †p<0.05, Tukey *p<0.05). FIG. 5O discloses anelectron micrograph of optic nerves of a control eye. FIG. 5P disclosesan electron micrograph of an OHT eye at low magnification. FIG. 5Qdiscloses an electron micrograph of AP of the OHT eye of FIG. 5P. FIG.5R discloses an electron micrograph of DAV of the OHT eye of FIG. 5P.FIG. 5S discloses an electron micrograph of an OHT eye treated withAlloP at low magnification. FIG. 5T discloses an electron micrograph ofAP of the OHT eye treated with AlloP of FIG. 5S. FIG. 5U discloses anelectron micrograph of AP of the OHT eye treated with AlloP of FIG. 5S.FIG. 5V discloses an electron micrograph of an OHT eye treated withent-AlloP at low magnification. FIG. 5W discloses an electron micrographof DAV of the OHT eye treated with ent-AlloP of FIG. 5V. FIG. 5Xdiscloses an electron micrograph of DAV of the OHT eye treated withent-AlloP of FIG. 5V. FIG. 5Y discloses the number of APs and FIG. 5Zdiscloses the number of DAVs *p<0.05.

FIGS. 6 (A-D) are exemplary embodiments of Western blotting in in vivoOHT eyes in accordance with the present disclosure. FIG. 6A disclosesWestern blot analysis of LC3. FIG. 6B discloses quantitative analysis ofLC3-II expression (n=4 per experiment, Dunnet †p<0.05, Tukey *p<0.05).*p<0.05. FIG. 6C discloses Western blot analysis of p62. FIG. 6Ddiscloses quantitative analysis of LC3-II expression (n=4 perexperiment, Dunnet †p<0.05, Tukey *p<0.05).

DETAILED DESCRIPTION OF THE DISCLOSURE

It is described herein that the neurosteroid, allopregnanolone (AlloP),and its enantiomer (ent-AlloP) are similarly neuroprotective in both invivo and ex vivo rat models of glaucoma. In the ex vivo model, ent-AlloPwas neuroprotective via autophagy activation but, unlike AlloP, not viaGAB AA receptor modulation. Immunoblotting analysis revealed that AlloPincreased LC3-II and decreased p62. Electron microscopic analysis showedthat AlloP increased autophagosomes (APs) without altering numbers ofdegenerative autophagic vacuoles (DAVs). ent-AlloP markedly increasedDAVs and LC3-II without altering APs but suppressing p62 levels moreeffectively, indicating that autophagy activation is a major mechanismunderlying its neuroprotective effects.

Thus, AlloP and ent-AlloP serve as potential therapeutic agents fortreatment of glaucoma but these enantiomers protect the retina bydistinct mechanisms. Therapeutic agents include compositions comprisingat least one of a neurosteroid and a synthetic enantiomer of theneurosteroid, such that an administered effective amount of thecomposition is a therapeutically effective amount. The exact amount of acompound required to achieve an effective amount will vary from subjectto subject, depending, for example, on age, general condition of asubject, identity of the particular compound(s), mode of administration,and the like. The compounds or neurosteroids of the present disclosureare useful for at least inducing autophagy, attenuating pressure-inducedretinal injury, and other benefits described herein in a subject, e.g.,a human subject, and are preferably administered in the form of aneffective amount of a compound (or combination of compounds) of theinstant disclosure and optionally or additional components.

In some embodiments, the present disclosure is directed to a compositionfor prevention or treatment of a neurodegenerative condition comprisinga neurosteroid, a synthetic enantiomer of a neurosteroid, or acombination thereof In some embodiments, the composition comprises aneurosteroid. In some embodiments, the composition comprises a syntheticenantiomer of a neurosteroid. In some embodiments, the compositioncomprises a neurosteroid and a synthetic enantiomer of a neurosteroid.

In some embodiments, the composition further comprises a salinesolution. Saline solutions are typically of physiological concentrationakin to extracellular fluid in the brain. In some embodiments, thecomposition further comprises (2-Hydroxypropyl)-β-cyclodextrin (2HBCD).2HBCD concentration in the composition is from about 1 millimolar toabout 10 millimolar, or about 20% (w/v).

In some embodiments, the neurosteroid is allopregnanolone and thesynthetic enantiomer of the neurosteroid is ent-allopregnanolone. Insome embodiments, the neurosteroid and enantiomeric neurosteroidincludes, but are not limited to, allopregnanolone, cholesterol,pregnenolone, progesterone, pregn-5α-ane-3,17-dione,pregn-5β-ane-3,17-dione, androsterone, etiocholanolone,tetrahydrodeoxycorticosterone, and any of the neurosteroids disclosed inU.S. Pat. No. 10,202,413 B2, which is herein incorporated by reference.

In some embodiments the neurosteroid, the synthetic enantiomer of theneurosteroid, or the combination thereof has a concentration of fromabout 10 nM to about 100 μM.

In some embodiments, the composition comprises the neurosteroid, and aconcentration of the neurosteroid is from about 10 nM to about 100 μM,from about 100 nM to about 10 μM, or about 1 μM.

In some embodiments, the composition comprises the synthetic enantiomerof the neurosteroid, and a concentration of the synthetic enantiomer isfrom about 10 nM to about 100 μM, from about 100 nM to about 10 μM, orabout 1 μM.

In some embodiments, the composition comprises both the neurosteroid andthe synthetic enantiomer of the neurosteroid, and a concentration of thecombination thereof is from about 10 nM to about 100 μM, from about 100nM to about 10 μM, or about 1 μM.

In some embodiments, the present disclosure is directed to a method ofpreventing or treating a neurodegenerative condition comprising inducingautophagy by administering an effective amount of the composition. Insome embodiments, the present disclosure is directed to a method ofattenuating pressure-induced retinal injury, the method comprisingadministering an effective amount of the composition.

In some embodiments, the neurodegenerative condition is selected fromglaucoma, age-related macular degeneration, retinal vascular occlusion,diabetic retinopathy, Alzheimer's disease and traumatic brain injury. Insome embodiments, the neurodegenerative condition is glaucoma.

In some embodiments, administering an effective amount of thecomposition comprises administering the composition by intravitrealinjection. In some embodiments, administering an effective amount of thecomposition comprises administering the composition by intracameralinjection.

EXAMPLES

Example 1. Effects of ent-AlloP on pressure-mediated retinaldegeneration in an ex vivo model—Retinas incubated at 10 mmHg (FIG. 1D)exhibited normal appearance but those at 75 mmHg showed axonal swellingin the nerve fiber layer (NFL) (FIG. 1E); this damage is attenuated by 1μM AlloP (FIG. 1F). Surprisingly, it was also found that 1 μM ent-AlloPsubstantially inhibited axonal swelling (FIG. 1G). To determine whetherthe neuroprotective effects of AlloP and ent-AlloP involve GABA_(A)receptors, 1 μM picrotoxin, a GABA_(A) receptor antagonist, wasadministered. Picrotoxin overcame the neuroprotective effect of AlloPunder hyperbaric conditions (FIG. 1H). However, picrotoxin did not alterthe effects of ent-AlloP (FIG. 1I), indicating that the mechanismunderlying its neuroprotection is distinct from GABA_(A) receptoractivation Table 1 (see also Table 2).

TABLE 1 Effects of AlloP and ent-AlloP on the NFLT, NDS, and density ofdamaged cells in the GCL. NFLT vs. RT (%) Damaged [p value vs. 75 NDScells in Condition mmHg] [p] GCL [p] 10 mmHg 1.5 ± 0.7 0.2 ± 0.4 1.9 ±1.8 75 mmHg 11.4 ± 1.9  0.9 ± 0.3 8.8 ± 2.2 [—] [—] [—] 75 mmHg + 1.5 ±0.6 0.2 ± 0.4 2.0 ± 1.7 1 μM AlloP [*p < 0.05] [p > 0.05] [*p < 0.05] 75mmHg + 1.6 ± 0.6 0.2 ± 0.4 1.9 ± 1.2 1 μM ent-AlloP [*p < 0.05] [p >0.05] [*p < 0.05] 75 mmHg + 4.0 ± 2.6 3.1 ± 0.8 23.9 ± 6.6  1 μM AlloP +[*p < 0.05] [*p < 0.05]  [*p < 0.05] 1 μM Picro 75 mmHg + 1 μM 1.5 ± 0.60.3 ± 0.5 2.1 ± 1.1 ent-AlloP + [*p < 0.05] [p > 0.05] [*p < 0.05] 1 μMPicro 75 mmHg + 1 μM 11.6 ± 0.8  1.4 ± 0.5 17.1 ± 6.0  ent-AlloP +  [p >0.05] [*p < 0.05]   [p > 0.05] 10 mM 3-MA

Data are mean±SD. NFLT vs. retinal thickness (RT) (%) refers to the NFLTpercentage of total RT. The density of damaged cells in the GCL wascounted per 250 μm of retina. P values in each parameter were calculatedby Dunnett's test.

TABLE 2 Morphological changes of retinas after pressure-loading andneurosteroids treatment. 75 mmHg + 75 mmHg + 75 mmHg + 1 μM 75 mmHg + 75mmHg + 1 μM 1 μM AlloP + ent-AlloP + 1 μM ent- 10 mmHg 75 mmHg 1 μMAlloP ent-AlloP 1 μM Picrotoxin 1 μM Picrotoxin AlloP + 3-MA NFLT 1 2.212.3  1.0 2.3 2.3 2.0 12.3  2 2.4 11.7  2.0 1.9 5.9 1.9 11.9  3 0.613.1  0.9 1.0 1.2 1.8 11.0  4 0.5 9.3 2.0 1.5 3.4 2.5 11.2  5 1.3 12.2 1.3 1.8 5.1 0.8 11    6 1.8 13.4  0.9 0.7 1.9 0.7 10.5  7 1.5 8.1 2.21.9 8.9 1.5 11.9  8 2   12.9  2.3 2.5 5.3 1.7 12.9  9 0.8 10.0  0.8 1.02.1 1.0 11.3  Average 1.5 11.4  1.5 1.6 4.0 1.5 11.6  SD 0.7 1.9 0.6 0.62.6 0.6 0.8 Dunnett's test vs  <0.05*  <0.05*  <0.05*  <0.05* >0.05 NDS1 0   1   1   0   4   0   1   2 1   1   0   1   3   0   2   3 0   0  0   0   2   0   1   4 0   1   0   1   4   1   1   5 0   1   0   0   4  0   1   6 1   1   0   0   3   1   2   7 0   0   1   0   3   0   2   80   1   0   0   2   1   1   9 0   1   0   0   3   0   2   Average 0.20.8 0.2 0.2 3.1 0.3 1.4 SD 0.4 0.4 0.4 0.4 0.8 0.5 0.5 Dunnett's testvs >0.05 >0.05  <0.05* >0.05  <0.05* Damaged cell 1 0   17    2   1  21    1   25    2 4   21    4   1   14    3   14    3 0   15    3   4  30    2   14    4 2   10    0   2   21    1   12    5 3   25    0   1  34    1   13    6 2   15    1   3   21    2   13    7 1   15    5   1  25    2   23    8 5   23    2   3   31    4   13    9 0   12    1   1  18    3   27    Average 1.9 17.0  2.0 1.9 23.9  2.1 17.1  SD 1.8 5.0 1.71.2 6.6 1.1 6.0 Dunnett's test vs  <0.05*  <0.05*  <0.05*  <0.05* >0.05

Example 2. ent-AlloP preserves neuronal nuclear antigen under highpressure—In whole mounted retinas, RGC damage induced by pressureelevation was visualized as a reduction in cells positive for NeuN. FIG.1J illustrates examples of confocal images of NeuN-labeled RGCs thatwere obtained from a control eye incubated at 10 mmHg. Pressureelevation (75 mmHg) reduced the number of cells positive for NeuN (FIG.1K). The confocal images in FIG. 1L and FIG. 1M illustrate theneuroprotective effects of AlloP (1 μM) and ent-AlloP (1 μM) on RGCsurvival in hyperbaric conditions, respectively. The preservation of RGCby ent-AlloP is as efficient as AlloP. The graph in FIG. 1N disclosesthe number of NeuN positive RGCs in the retina in each condition (Table3).

TABLE 3 RGC Survival (FIG.1N) 75 mmHg + 75 mmHg + 10 mmHg 75 mmHg AlloPent-AlloP 1 2500 480 2240       2280       2 2060 840 1860      2000       3 1980 460 2260       1840       4 2180 540 1840      1860       5 2160 480 2220       2120       Average 2176 560.02084.0     2020.0     SD 198.2 159.4 214.2    184.4    Dunnett vs <0.05*<0.05* Tukey vs <0.05* <0.05* vs >0.05 

Example 3. ent-AlloP prevents pressure-induced apoptosis—At 10 mmHg, afew TUNEL-positive cells are observed in the ganglion cell layer (GCL)and outer nuclear layer (ONL) (FIG. 1O). Exposure to elevated pressure(75 mmHg) induced apoptosis that was apparent in the GCL and the innernuclear layer (INL) (FIG. 1P). The number of TUNEL-positive cells wasreduced by AlloP (FIG. 1Q). Similar protection was observed withent-AlloP (FIG. 1R). TUNEL staining with 1 μM AlloP (FIG. 1Q) or 1 μMent-AlloP (FIG. 1R) showed significant decrease in the number ofTUNEL-positive cells at 75 mmHg. The graph in FIG. 1S discloses thenumber of apoptotic cells in the retina in each condition (Table 4).

TABLE 4 TUNEL Staining (FIG. 1S) 75 mmHg + 75 mmHg + 10 mmHg 75 mmHgAlloP ent-AlloP 1 3 62 0    1    2 3 60 0    0    3 0 57 0    0    4 626 2    2    5 2 49 7    10     Average 2.8 50.8 1.8  2.6  SD 2.2 14.73.0  4.2  Dunnett vs <0.05* <0.05* Tukey vs <0.05* <0.05* vs >0.05 

Example 4. Autophagy vacuoles induced by high pressure and effects ofneurosteroids—Because AlloP activates autophagy in primary astrocytecultures, the activation of autophagy induced by AlloP and ent-AlloP wasexamined. Electron microscopy revealed that retinas incubated at 10 mmHgremained intact (FIG. 2A), while substantial swelling of axons wasobserved after exposure to high pressure (75 mmHg; FIG. 2B). Similar toAlloP (FIG. 2C), ent-AlloP (FIG. 2D) protected RGC axons from highpressure. Retinas were then examined for the presence of autophagosomes(APs) and degenerative autophagic vacuoles (DAVs). These structures arepresent even at control pressure (10 mmHg; FIG. 2E-G). Elevated pressuresignificantly increased numbers of APs and other autophagy components inthe NFL (FIG. 2H-J). The increase in APs was robustly altered by AlloP,without effect on DAVs (FIG. 2K, 2L). Interestingly, the highpressure-mediated increase in APs was significantly decreased byent-AlloP, while DAVs in the NFL were significantly increased (FIG. 2M).A quantitative assessment of APs and DAVs induced by pressure elevationand administration of AlloP or ent-AlloP is summarized in FIG. 2N andFIG. 2O, respectively (Table 5, 6).

TABLE 5 Autophagosomes (AP)/25 μm2 in the NFL (FIG. 2N) 10 mmHg 75 mmHgAlloP ent-AlloP 1 0 0 1    0   2 0 0 2    0   3 1 0 2    0   4 0 1 0   1   5 0 0 2    0   6 0 0 0    0   7 0 0 2    0   8 0 0 2    0   9 0 03    0   10 0 0 2    0   Total 1 1 16     1   Average 0.1 0.1 1.6  0.1SD 0.3 0.4 0.9  0.4 Dunnett vs p > 0.05 <0.05* >0.05 vs p > 0.05<0.05* >0.05 Tukey vs <0.05* >0.05 vs  <0.05*

TABLE 6 Degenerative autophagic vacuoles (DAV)/ 25 μm2 in the NFL (FIG.2O) 10 mmHg 75 mmHg AlloP ent-AlloP 1 0 0   3   3    2 0 0   1   1    30 0   0   4    4 0 1   1   1    5 1 0   0   1    6 1 0   0   2    7 01   1   2    8 0 0   1   2    9 0 0   0   2    10 0 2   1   4    Total 24   8   22     Average 0.2 0.4 0.8 2.2  SD 0.4 0.4 1.2 1.4  Dunnettvs >0.05 >0.05 <0.05* vs >0.05 >0.05 <0.05* Tukey vs >0.05 <0.05* vs<0.05*

Example 5. Autophagy markers and effects of ent-AlloP on autophagyflow—Microtubule-associated protein-1 light chain 3 (LC3) is a corecontributor to autophagy (FIG. 1A), playing a crucial role in elongationof phagophore membranes and serving as a marker of autophagy. LC3antibodies displayed double bands (LC3-I and LC3-II) at approximately14-16 kDa. Quantitative Western blot analysis demonstrated thatadministration of both neurosteroids significantly increased LC3-IIexpression compared to drug-free pressure elevation (FIG. 2P and FIG.2Q, Table 7). Because both LC3-I and LC3-II were increased by bothneurosteroids, it was necessary to examine another autophagy marker toassess changes in autophagic flow. P62 is a protein that is incorporatedinto completed autophagosomes and accumulates when autophagy isimpaired. Although Western blot analysis demonstrated that AlloP alteredexpression of p62 compared to controls incubated at 75 mmHg, ent-AlloPmore effectively depressed p62 compared to AlloP (FIG. 2R, 2S, Table 8).

TABLE 7 LC3-II expression (FIG.2Q) Control OHT AlloP ent-AlloP 1 1.00 1.50 4.31 6.14 2 1.00  2.03 3.47 5.17 3 1.00  1.23 3.34 4.48 4 1.00 2.15 3.34 5.22 Average 1.00  1.73 3.62 5.25 SD 0.02  0.45 0.49 0.70Dunnett vs >0.05 <0.05* <0.05* Tukey vs >0.05 <0.05* <0.05* vs <0.05*<0.05* vs <0.05*

TABLE 8 p62 expression (FIG.2S) 10 mmHg 75 mmHg AlloP ent-AlloP 1 1.003.30  1.96 0.92 2 1.00 3.16  2.18 0.40 3 1.00 4.89  2.05 1.00 4 1.002.08  0.84 0.28 5 1.00 3.12  2.81 1.08 Average 1.00 3.31  1.97 0.74 SD0.02 1.18  0.64 0.38 Dunnett vs <0.05* >0.05 >0.05  Tukey vs<0.05* >0.05 >0.05  vs  <0.05* <0.05* vs <0.05*

Example 6. 3-MA blocks autophagy and inhibits the neuroprotectiveeffects of ent-AlloP—To determine whether autophagy plays a key role inretinal protection by ent-AlloP 3-methyladenine (3-MA), an inhibitor ofautophagic flux, was examined. At 10 mM, 3-MA, did not alterneuroprotection by AlloP (FIG. 3A), but dampened the effects ofent-AlloP at 75 mmHg (FIG. 3B). It was also found that 3-MA alone wasneurodegenerative at both 75 mmHg (FIG. 3C) and 10 mmHg (FIG. 3D),indicating that autophagy is important for maintaining retinal integrityeven under control conditions. A combination of ent-AlloP and 3-MAsignificantly increased the density of damaged cells in the GCL comparedto controls incubated at 75 mmHg (p<0.05) and overcame the protectiveeffects of ent-AlloP at high pressure (Table 1, Table 2). It was alsoexamined whether 3-MA altered the effects of ent-AlloP on autophagicmarkers, and it was found that upregulation of LC3-II levels induced byent-AlloP was dampened by 3-MA (FIG. 3E, 3F, Table 9). Also, thedecrease of p62 levels induced by ent-AlloP at 75 mmHg was reversed by3-MA (FIG. 3G, 3H, Table 10). Administration of 10 mM 3-MA significantlyincreased LC3-II expression in the retina incubated with ent-AlloP at 75mmHg (Wilcoxon Rank-Sum Test, *p<0.05), as shown in FIG. 3 (A-H). Thesefindings indicate that ent-AlloP likely acts by enhancing autophagicflow.

TABLE 9 LC3 expression after 3-MA treatment (FIG. 3F) ent-AlloPent-AlloP + 3-MA 1 11.20 1.00 2 8.89 1.00 3 7.78 1.00 4 8.11 1.00Average 9.00 1.00 SD 1.54 0.00 Wicoxon vs p = 0.0139

TABLE 10 p62 expression after 3-MA treatment (FIG. 3H) ent-AlloPent-AlloP + 3-MA 1 1.00 3.21 2 1.00 2.11 3 1.00 1.76 4 1.00 1.66 Average1.00 2.19 SD 0.00 0.71 Wicoxon MW vs p = 0.0139

Example 7. Rat in vivo OHT model induced by intracameral injection ofmicrobeads—For induction of ocular hypertension (OHT) in vivo, sterile 6μm polystyrene microbeads were injected into the anterior chamber of theleft eye of each animal using a single-step, sclero-corneal tunnelapproach with a 35G nanoneedle (FIG. 4A). At 3 weeks after intracameralbead injection, the beads were localized in the iridocorneal angle,especially in the inferior area (FIG. 4B and 4C). After stabilization ofelevated IOP, animals were randomly divided into 3 groups (non-treatedOHT, AlloP injection, and ent-AlloP injection). AlloP and ent-AlloP wereadministered as a one-time intravitreal injection one week followingbead injection. Non-treated OHT animals received sterile vehicleintravitreally. As a further control, vehicle was intravitreallyinjected 7 days after intracameral administration of 10 μl of PBS. Threeweeks after bead injections, IOP was 26.7±3.7 mmHg in non-treated OHTeyes compared to 9.9±0.9 mmHg in control eyes. At each measurement timepoint (days 3, 7, 14, and 21), IOP in the three bead-injected groups(non-treated OHT, AlloP injection, and ent-AlloP injection) wassignificantly higher compared to control eyes (p<0.05, WilcoxonMann-Whitney test) (FIG. 4D, Tables 11A-D). The neurosteroids did notalter TOP at any measurement point.

TABLE 11A IOP profile, AlloP (FIG. 4D) AlloP Pre-ope 3 days 7 days 14days 21 days 1 10 25 29 33 30 2 11 31 33 36 25 3 10 27 41 31 24 4 11 2536 31 33 5 12 31 35 32 31 6 9 25 30 25 24 7 10 27 25 21 26 8 10 28 21 1927 9 11 20 32 30 21 10 10 25 21 27 33 11 10 28 31 28 31 12 10 22 25 2530 13 9 33 23 20 28 14 9 30 20 36 25 Average 10.1 26.9 28.7 28.1 27.7 SD0.9 3.6 6.4 5.6 3.7 Dunnett vs p < 0.05* p < 0.05* p < 0.05* p < 0.05*

TABLE 11B IOP profile, ent-AlloP (FIG. 4D) ent-AlloP Pre-ope 3 days 7days 14 days 21 days 1 9 22 25 31 22 2 8 32 21 20 27 3 10 27 38 35 36 412 20 39 41 34 5 13 32 27 25 30 6 10 25 21 22 21 7 9 33 26 21 24 8 8 3623 20 33 9 10 28 21 35 31 10 10 20 31 29 20 11 10 25 34 20 33 12 9 15 4231 30 13 11 30 32 33 35 14 12 23 37 32 29 Average 10.1 26.3 29.8 28.228.9 SD 1.5 6.0 7.3 6.9 5.3 Dunnett vs p < 0.05* p < 0.05* p < 0.05* p <0.05*

TABLE 11C IOP profile, OHT (FIG. 4D) OHT (vehicle control) Pre-ope 3days 7 days 14 days 21 days 1 10 21 31 29 28 2 8 26 30 30 20 3 10 29 2930 31 4 11 30 21 20 25 5 10 22 19 19 28 6 10 21 30 30 30 7 10 14 29 2825 8 9 30 25 20 35 9 9 21 21 27 28 10 10 25 34 21 20 11 10 25 31 20 3312 10 38 30 31 30 13 11 30 32 33 35 14 10 21 29 32 29 Average 9.9 23.327.0 26.4 26.7 SD 0.9 5.5 4.9 5.2 3.7 Dunnett vs p < 0.05* p < 0.05* p <0.05* p < 0.05*

TABLE 11D IOP profile, intact control (FIG. 4D) Intact Pre- control ope3 days 7 days 14 days 21 days 1 9 10 11 9 8 2 8 10 10 10 10 3 10 12 9 1011 4 11 11 11 10 10 5 10 10 9 10 10 6 10 9 10 11 10 7 10 11 9 8 10 8 910 10 11 12 9 10 9 13 11 10 10 10 9 10 9 10 11 10 10 9 9 10 12 9 12 1112 13 13 11 11 10 10 11 14 12 13 9 8 8 Average 9.7 10.4 9.9 9.9 9.9 SD1.0 1.0 0.9 1.2 0.9 Dunnett vs p < 0.05* p < 0.05* p < 0.05* p < 0.05*

Example 8. Pressure-induced apoptosis and neuroprotection with AlloP andent-AlloP in vivo—Three weeks after intracameral administration of PBS,a few TUNEL-positive cells are observed in the INL and ONL in controleyes (FIG. 4E). In contrast, apoptosis was markedly increased in theGCL, INL, and ONL in non-treated OHT eyes (FIG. 4F). The number ofTUNEL-positive cells was reduced to control levels when 1 μM AlloP (FIG.4G) or 1 μM ent-AlloP (FIG. 4H) was administered intravitreally in theOHT eyes. The graph in FIG. 4I discloses the number of apoptotic cellsin the retina in each condition (Table 12).

TABLE 12 TUNEL staining (FIG. 4I) Control OHT AlloP ent-AlloP 1 1 30 1 12 1 30 0 0 3 0 32 1 0 4 2 21 0 0 5 10 16 0 1 Average 2.8 25.8 0.4 0.4 SD4.1 6.9 0.5 0.5 Dunnett vs p < 0.05* p < 0.05* Tukey vs p < 0.05* p <0.05* vs p < 0.05*

Example 9. RGC survival by neurosteroids during IOP elevation—In wholemounted retinas, glaucomatous damage was visible as reduced numbers ofcells that were positive for NeuN three weeks after microbead injectionin non-treated OHT eyes compared with control eyes (FIG. 4J, 4K).Administration of AlloP (FIG. 4L) or ent-AlloP (FIG. 4M) prevented theloss of NeuN-positive RGCs in OHT eyes. RGC density at each pressure issummarized in FIG. 4N (Table 13). In sections of optic nerves stainedwith 2% toluidine blue, axonal loss was detected 3 weeks after microbeadinjection in non-treated OHT eyes compared with control eyes (FIG. 4O, 4p). However, administration of AlloP (FIG. 4Q) or ent-AlloP (FIG. 4R)induced significant protective effects in OHT eyes. The density of axonsin each experiment is summarized in FIG. 4S (Table 14).

TABLE 13 RGC Survival (FIG.4N) Control OHT AlloP ent-AlloP 1 2420 10202200 2060 2 2840 1240 2240 2260 3 1920 780 1940 2500 4 2060 840 18602200 5 2020 900 1800 2460 Average 2252.0 956.0 2008.0 2296.0 SD 379.1181.9 200.3 183.5 Dunnett vs p < 0.05* p < 0.05* Tukey vs p < 0.05* p <0.05* vs p < 0.05*

TABLE 14 Density of axons in ONs (Axons × 10000/ square mm) (FIG. 4S)Control OHT AlloP ent-AlloP 1 29 16 27 33 2 33 18 32 32 3 32 15 29 29 435 21 33 27 5 28 19 33 31 Average 31.4 17.8 30.8 30.4 SD 2.9 2.4 2.7 2.4Dunnett vs p < 0.05* p < 0.05* Tukey vs p < 0.05* p < 0.05* vs p < 0.05*

Example 10. Autophagy vacuoles by IOP elevation and effects ofneurosteroids—Electron microscopy revealed the presence of APs and DAVsin the NFL of control eyes (FIG. 5A, 5B, 5C) and OHT eyes (FIG. 5D, 5E,5F). AlloP significantly increased the number of APs in the NFL in OHTeyes (FIG. 5G, 5H, 5I), while ent-AlloP significantly decreased thenumber of APs compared to AlloP-treated OHT eyes (FIG. 5J, 5K, 5L). DAVsin the NFL were significantly increased by 1 μM ent-AlloP compared tonon-treated OHT eyes. A quantitative assessment of APs and DAVs inducedby OHT and administration of AlloP or ent-AlloP is summarized in FIG. 5Mand FIG. 5N, respectively (Table 15, 16).

TABLE 15 Autophagosomes (AP)/25 μm2 in the NFL (FIG. 5M) Control OHTAlloP ent-AlloP 1 0 1 1 0 2 0 0 1 0 3 0 0 1 0 4 0 0 1 0 5 0 0 2 1 6 0 12 0 7 0 0 1 0 8 0 0 1 0 9 0 0 1 0 10 0 1 1 0 Total 0 3 12 1 Average 0.00.3 1.2 0.1 SD 0.0 0.4 0.4 0.4 Dunnett vs p > 0.05 *p < 0.05 p > 0.05Tukey vs p > 0.05 *p < 0.05 p > 0.05 vs  p < 0.05 p > 0.05 vs *p < 0.05 

TABLE 16 Degenerative autophagic vacuoles (DAV)/ 25 μm2 in the NFL (FIG.5N) Control OHT AlloP ent-AlloP 1 0 1 1 4 2 0 2 0 4 3 0 0 0 3 4 0 0 0 05 1 0 0 1 6 0 1 2 2 7 0 0 0 2 8 0 1 2 2 9 0 1 0 2 10 0 0 0 2 Total 1 6 522 Average 0.1 0.6 0.5 2.2 SD 0.3 0.9 0.4 1.8 Dunnett vs p > 0.05 p >0.05 *p < 0.05 Tukey vs p > 0.05 p > 0.05 *p < 0.05 vs p > 0.05 *p <0.05 vs *p < 0.05

APs and DAVs were also observed in the optic nerves (ON) of control eyes(FIG. 5O). Compared to non-treated OHT eyes (FIG. 5P, 5Q, 5R), thenumber of APs significantly increased in the ON of OHT eyes (FIG. 5S,5T, 5U), while ent-AlloP significantly decreased APs (FIG. 5V, 5W, 5X).DAVs in the ON were significantly increased by administration of 1 μMent-AlloP compared to non-treated OHT eyes. A quantitative assessment ofAps and DAVs in the ON induced by OHT and administration of AlloP orent-AlloP is summarized in FIG. 5Y and FIG. 5Z, respectively (Table 17,18).

TABLE 17 Autophagosomes (AP)/100 μm2 in the ON (FIG. 5Y) Control OHTAlloP ent-AlloP 1 0 0 1 0 2 0 1 1 1 3 0 0 1 0 4 0 0 1 0 5 0 0 2 0 6 0 11 0 7 0 0 1 0 8 0 0 1 0 9 0 0 1 0 10 0 1 1 0 Average 0.0 0.3 1.1 0.1 SD0.0 0.4 0.4 0.4 Dunnett vs p > 0.05 *p < 0.05 p > 0.05 Tukey vs p > 0.05*p < 0.05 p > 0.05 vs *p < 0.05 p > 0.05 vs *p < 0.05 

TABLE 18 Degenerative autophagic vacuoles (DAV)/ 100 μm2 in the ON (FIG.5Z) Control OHT AlloP ent-AlloP 1 0 0 0 1 2 0 0 0 1 3 0 1 0 1 4 0 0 0 25 1 0 0 0 6 0 0 0 1 7 0 0 0 1 8 0 0 0 1 9 0 0 0 1 10 0 0 0 1 Average 0.10.1 0.0 1.0 SD 0.3 0.4 0.0 0.7 Dunnett vs p > 0.05 p > 0.05 *p < 0.05Tukey vs p > 0.05 p > 0.05 *p < 0.05 vs p > 0.05 *p < 0.05 vs *p < 0.05

Example 11. Autophagy markers after IOP elevation and effects ofneurosteroids—Quantitative Western blot analysis demonstrated that thelevel of LC3-II significantly increased in OHT eyes compared to controleyes three weeks after microbead injection (FIG. 6A, 6B, Table 19). Inaddition, 1 μM ent-AlloP significantly increased the level of LC3-IIcompared to OHT eyes treated with AlloP or OHT without neurosteroidadministration 3 weeks after microbead injection (FIG. 6A, FIG. 6B,Table 19). Western blotting also demonstrated that p62 was significantlyincreased in eyes with OHT compared with control eyes 3 weeks aftermicrobead injection (FIG. 6C, 6D, Table 20). Administration of 1 μMAlloP or 1 ent-AlloP significantly depressed the level of p62 comparedto OHT eyes without neurosteroid administration (FIG. 6D, Table 20).

TABLE 19 LC3-II expression (FIG. 6B) 10 mmHg 75 mmHg AlloP ent-AlloP 11.00 1.36 2.73 4.96 2 1.00 2.00 3.32 4.97 3 1.00 1.53 2.82 4.27 4 1.001.77 3.11 5.04 Average 1.00 1.67 3.00 4.81 SD 0.02 0.30 0.29 0.38Dunnett vs <0.05* <0.05* <0.05* Tukey vs <0.05* <0.05* <0.05* vs <0.05*<0.05* vs <0.05*

TABLE 20 p62 expression (FIG. 6D) Control OHT AlloP ent-AlloP 1 1.002.86 1.73  0.92 2 1.00 2.40 1.82  0.91 3 1.00 4.07 2.90  1.00 4 1.002.96 2.53  1.61 Average 1.00 3.07 2.25  1.11 SD 0.02 0.73 0.58  0.35Dunnett vs <0.05* <0.05* p > 0.05 Tukey vs <0.05* <0.05* p > 0.05vs >0.05   <0.05* vs  <0.05*

Discussion

These results demonstrate the therapeutic effect of AlloP enantiomersand the involvement of autophagy as a protective mechanism in glaucoma.Although apoptosis is believed to be a mechanism that kills RCGs inglaucoma, autophagy may serve either a neurotoxic or neuroprotectiverole. Indeed, autophagy often coexists with apoptosis in glaucomamodels. Eight weeks after IOP elevation, an increase in LC3-II/I ratiowas noted in rat RGCs. Because LC3-II is associated with autophagosomeformation, the increased ratio indicates induction of autophagy.Autophagic vacuoles were also detected in axons of rat optic nerves 3weeks after IOP elevation.

Similarly, autophagy in RGCs has been observed in a rhesus monkey OHTglaucoma model. In an ex vivo glaucoma model, apoptotic RGCs wereobserved along with modest increases in DAVs and an increase in LC3-IIconsistent with autophagy induction. (FIG. 5M-N and FIG. 6B). Thesechanges were manifest by 24 hours after pressure elevation, consistentwith findings that autophagy induction in RGCs occurs soon after acuteaxonal injury. It remains uncertain whether autophagy in RGCs isneuroprotective or neurodegenerative. While apoptosis is described astype I programed cell death (PCD), autophagy is considered to be a typeII PCD, possibly contributing to RGC death. Autophagy activation in RGCsresults in apoptosis, suggesting that autophagy is neurodegenerative.Similarly, in a model of dementia, inhibition of early autophagy with3-MA is neuroprotective, suggesting that autophagy contributes toneuronal damage. However, the primary role of autophagy is to promotecell survival by recycling cellular debris, resulting inneuroprotection, as observed in a mouse model of optic nervetransection.

Similarly, in a rat chronic hypertensive model, rapamycin, an activatorof autophagy, aids RGC survival. Rapamycin also protects RGCs fromdamage induced by ROS. 3-MA was found to be neurodegenerative even incontrols, indicating that inhibiting autophagy can have deleteriouseffects in the retina, and 3-MA induced degeneration of RGC was observedat both high (FIG. 3C) and low pressure (FIG. 3D).

As disclosed herein, the enantiomers of AlloP were similarlyneuroprotective in both ex vivo and in vivo OHT models. AlloP andent-AlloP are mirror image molecules that have markedly differenteffects on GABA_(A) receptors. Consistent with this, it was found thatpicrotoxin, an inhibitor of GABA_(A) receptors, discriminates betweenthe neuroprotection of these enantiomers. While picrotoxin prevents theneuroprotective effects of AlloP at high pressure (FIG. 1H), it did notaffect neuroprotection by ent-AlloP (FIG. 1I), indicating thatneuroprotection by ent-AlloP is distinct from GABA_(A) receptors. Thisobservation led to consideration of alternative mechanisms forent-AlloP.

In electron microscopic (TEM)-based analyses, ent-AlloP decreased APsbut robustly increased DAVs in the NFL, indicating activation ofautophagy flow. Although AlloP had partial effects on the increase inAPs, AlloP failed to increase DAVs. These observations indicate thatent-AlloP exerts axonal protection via autophagy activation moreeffectively than AlloP, and also indicate that activation of autophagyby high pressure alone, without AlloP or ent-AlloP, is insufficient toprotect RGCs from high pressure-driven damage.

These observations were confirmed with LC3 and p62 analyses. Becausecytosolic LC3-I is conjugated to phosphatidyl ethanolamine to formLC3-II, LC3-II can be increased even if autophagy flow is not fullyactivated, consistent with the increase in LC3-II that was observed withAlloP. However, the effects of ent-AlloP are more prominent than thoseof AlloP (FIG. 2R-S). To determine the involvement of autophagy inneuroprotection by ent-AlloP, the effects of 3-MA on LC3 proteins wereexamined. 3-MA blocks autophagy by inhibiting class IIIphosphatidylinositol 3-kinase (PtdIns3K), an enzyme required for themembrane dynamics involved in autophagic vesicle trafficking, thuspreventing formation of autophagosomes. It was found that 3-MAattenuated LC3-II upregulation (FIG. 3D) induced by ent-AlloP. p62levels inversely correlate with autophagy activity, and when autophagyis inhibited p62 accumulates. Under pathological conditions, there is aconstitutively high level of p62, leading to accumulation of damagedmitochondria and subsequent ROS production. Thus, the reduction of p62expression by ent-AlloP (FIG. 2R) and the enhancement by 3-MA (FIG. 3E)indicate again the effective activation of autophagy flow by ent-AlloP.In the ex vivo glaucoma model described herein, it is concluded thatautophagy induced by high pressure alone is not sufficient to preventapoptosis. However, ent-AlloP successfully promotes autophagy andprevents apoptosis in high pressure.

Using a rat in vivo OHT glaucoma model, the neuroprotective effects ofent-AlloP via autophagy were further demonstrated. As in the ex vivomodel, promotion of RGC survival and protection against apoptosis in OHTare similar between AlloP and ent-AlloP. However, ent-AlloP decreased APnumbers but robustly increased DAV numbers in the NFL and ON, whileAlloP increased AP numbers, but did not alter DAVs. These findingsindicate again that ent-AlloP activates autophagy flow more effectivelythan AlloP, and a significant increase in LC3-II with high pressure isshown. Additionally, ent-AlloP increased levels of LC3-II and decreasedp62 in the OHT retinas.

From these observations, it is concluded that autophagy occurring in OHTis neuroprotective rather than neurodegenerative, but, in the absence ofaugmentation by ent-AlloP, pressure-driven autophagy is insufficient toprotect the retina. It is also concluded that both AlloP and ent-AlloPserve as potential therapeutic agents for treatment of glaucoma but thatthese enantiomers act by distinct mechanisms, providing uniqueadvantages to one or the other enantiomer in different clinicalsituations. The compounds or neurosteroids of the present disclosure areuseful for at least inducing autophagy, attenuating pressure-inducedretinal injury, and other benefits described herein in a subject, e.g.,a human subject, and are preferably administered in the form of aneffective amount of a compound (or combination of compounds) of theinstant disclosure and optionally or additional components. Theseresults also have implications for the development of neurosteroids astreatments for other neurodegenerative disorders.

Materials and Methods—Protocols for animal use were approved by theAkita University Animal Studies Committee in accordance with theguidelines of the Policies on the Use of Animals and Humans inNeuroscience Research.

Rat ex vivo Eyecup Preparation—Rat ex vivo eyecups were prepared from28-32 day old male Sprague-Dawley rats (Charles River LaboratoriesInternational Inc., Wilmington, MA). The anterior half of the enucleatedeyes was carefully removed to make eyecup preparations. Eyecups wereplaced at the bottom of a 100 ml glass beaker filled with aCSF(artificial cerebrospinal fluid) containing (in mM): 124 NaCl, 5 KCl, 2MgSO₄, 2 CaCl₂, 1.25 NaH₂PO₄, 22 NaHCO₃, and 10 glucose, and incubatedat 30° C. for 24 hours using a closed pressure-loading system (FIG. 1C).pH was maintained at 7.35 to 7.40. In the closed-pressure system, aglass beaker with the eyecup was placed at the bottom of an acrylicpressure chamber (2,000 ml volume). A 95% O₂-5% CO₂ gas mixture wasdelivered through disposable plastic tubing with an infusion valve and acontrol dial on the lid of the pressure chamber and an air filter (Cat#SLGP033RS, Merck Millipore, Billerica, MA). The plastic tubingdelivering the gas terminated 1 cm above the bottom of the beaker.

Acutely prepared eyecups were incubated in gassed aCSF for at least 1 hat 30° C. before pressure loading. In some experiments, AlloP (1 μM),ent-AlloP (1 μM), and 3-MA (10 mM) were dissolved in aCSF at the time ofexperiment and administered by bath perfusion. Eyecup preparations weretreated with these drugs for 1 h at 30° C. before pressure loading. Forpressure loading, the 95% O₂-5% CO₂ gas mixture was infused until thepressure reading given by a manometer reached the appropriate level. Thepressure was then locked by adjusting the control dial of the effusionvalve, and monitored continuously for 24 h at 30° C. After maintainingthe chamber at the set pressure (10 mmHg and 75 mmHg) for the indicatedtime, the pressure inside the chamber was carefully decreased by openingthe effusion valve.

Rat in vivo OHT model—8-week old male Sprague-Dawley rats were deeplyanesthetized with an intraperitoneal injection of a mixture ofmedetomidine hydrochloride (Cat #133-17474, CAS. No 86347-15-1, WakoPure Chemical Industries Ltd., Osaka, Japan, 0.15 mg/kg), midazolam (Cat#135-13791, CAS. No 58786-99-5, Wako Pure Chemical Industries Ltd., 2mg/kg), and butorphanol tartrate (Cat #021-19001, CAS. No 86347-15-1,Wako Pure Chemical Industries Ltd., 2.5 mg/kg), and intraocular pressurewas increased unilaterally to approximately 35 mmHg, by intracameralinjection of polystyrene microbeads using a single-step, sclero-cornealtunnel approach with a 35G-gauge nanoneedle (#LNAN-3505LM, Saito MedicalInstruments Inc., Tokyo, Japan) connected to 100 μl WPI Nanofil 100microsyringe (World Precision Instruments, Inc., Sarasota, FL). Sterile6 μm polystyrene microbeads in a 1×10⁶ microbeads/ml solution (MolecularProbes, Eugene, OR, USA) were concentrated to obtain a 1×10⁷microbeads/ml solution. This solution was suspended in PBS. Themicrobeads suspended in PBS were aspirated into a 100 μl Hamiltonmicrosyringe and injected into the anterior chamber of right eye of eachanimal. 10 μl of the microbead suspension were injected withoutcontacting the cornea or iris with the needle. After stabilization ofthe elevated IOP, animals were randomly divided into 3 groups(non-treated OHT, AlloP injection, and ent-AlloP injection). AlloP andent-AlloP were dissolved in 20% w/v (2-Hydroxypropyl)-β-cyclodextrin(2HBCD) in saline solution at concentration of 0.05% (w/v) solution, andinjected into the vitreous chamber in a total volume of 1 μl using aHamilton syringe adapted with a 35 G-gauge nanoneedle 1 week after thebeads were injected under halothane inhalation anesthesia. OHT animals(vehicle control) received sterile 1 μl 20% (w/v) 2HBCD with PBS. As afurther control, 1 μl 20% (w/v) 2HBCD was intravitreally injected 7 daysafter intracameral injection with 10 μl of PBS. The number of animalsused in each experiment is indicated in the corresponding method. Thetip of the needle was inserted into the superior hemisphere of the eyeat a 45° angle through the sclera into the vitreous body to avoidretinal detachment or injury to eye structures. Ocular hypertension wasmonitored preoperatively, and at 3 days, 1 week, 2 weeks or 3 weeksafter the beads were injected. IOP was measured using a laboratorytonometer (TonoLab, Icare, Finland).

Electron Microscopy—Retinal specimens were trimmed to a smaller size,and ultrathin sections (75 nm) were cut with a diamond knife andsuspended over formvar-coated slot grids (1×2 mm opening). The sectionswere stained with uranyl acetate and lead citrate and viewed in atransmission electron microscope (H-7650, Hitachi High-TechnologiesCorp., Tokyo, Japan). Numbers of AP and DAV inside NFLs and axons weredetermined as the sum in 10 different areas of 25 μm² each from eachsample. The analysis was performed in three eyes per experimentalcondition.

Data analysis of morphometry—The middle portion of the retina wasexamined, greater than 1,200 μm away from the center of the optic discalong the inner limiting membrane (ILM). The nerve fiber layer thickness(NFLT) was measured by light microscopy along 5 lines perpendicular tothe pigment epithelium at a distance of 15 μm from each other around1,200 μm away from the center of the optic disc. The average NFLT wasdetermined in 9 different light micrographs taken from 5 eyecup samplesin each condition, divided by total retinal thickness, and mean±standarddeviation (%) was analyzed and compared with control.

The density of degenerated cells characterized by nuclear chromatinclumping or necrosis in the GCL was determined by counting 9 fields of500 μm length in light micrographs taken from the block of the middleretinal part 950 to 1450 μm away from the center of the optic disc.

The severity of neuronal damage was assessed by light microscopy using aneuronal damage score (NDS). The NDS was determined in 9 different lightmicrographs taken from 5 eyecup samples in each condition. The NDS ratesneuronal damage in the inner nuclear layer (INL) and the inner plexiformlayer (IPL) on a 0-4 scale with 0 signifying no neuronal damage and 4indicating very severe damage. Criteria used in establishing the degreeof neuronal damage included the extent of cytoplasmic swelling in theIPL and the number of neurons in the INL showing signs of severecytoplasmic swelling and coarse clumping of nuclear chromatin. Thehighest NDS rating (4) is given when the IPL discloses apparentspongiform appearance due to dendritic swelling and when most cellbodies in the INL show severe cytoplasmic swelling and coarse clumpingof nuclear chromatin. If the damage is of a lesser degree, a rating of 3is given. NDS 2 is assigned when cell bodies in the INL are sporadicallyswollen. In NDS 1, damage does not fulfill higher criteria but theretinas differ from controls (NDS 0). Fine dendritic swelling in alimited area of the IPL without damage in the INL is described by NDS 1.

NFLT, density of degenerated cells in the GCL, and NDS were determinedas the measurement of 10 different areas from three eyes perexperimental condition. These morphometrical parameters were assessed bythree raters, who remained unaware of the experimental condition. Uponcompletion of data assessment, significance of individual differencesamong raters was evaluated using five randomly selected samples in eachmorphometric parameter by one-way analysis of variance (one-way ANOVA)followed by a post-hoc test. There were no significant differences amongthe raters in any of the morphometric measurements. All data of the NFLT(% vs. retinal thickness), NDS, and the density of degenerated cells inthe GCL are expressed as mean±SEM, and evaluated by Dunnett's multiplecomparison test compared to controls incubated in aCSF at 10 mmHg or 75mmHg

Preparation of whole mounted retinas and immunostaining—The retina wascarefully detached from the eye by making cuts along the ora serrata andoptic nerve. Whole retinas were then flat-mounted, pinned in an acrylicplate with the RGC layer facing upward using stainless steel pins, andfixed in 4% paraformaldehyde-0.1 M phosphate buffer overnight at 4° C.After the samples were fixed, the tissue was rinsed with PBS threetimes. To block nonspecific binding, the tissue was incubated in 2% BSAin PBS containing 0.5% Triton X-100. The whole mounted retinas wereincubated in the rabbit anti-NeuN polyclonal antibody solution (Cat#ab104225, Abcam, Cambridge, MA)) (1:100) by gently shaking at 4° C.,overnight. After rinsing 3 times using PBS, the retina was incubated inFITC-conjugated secondary antibody (goat anti-rabbit IgG (H+L)) (Cat#81-6111, Zymed Laboratories Inc., San Francisco, CA) (1:300). Theretina tissue was then rinsed 3 times with PBS and mounted on glassslides using 50% PBS and 50% glycerol. Retinal flat-mounts were imagedthroughout the GCL in each of the four defined retinal quadrants 4 mmfrom the optic nerve head using a confocal microscope. Each quadrant wasanalyzed using a 1 mm² frame, and counted using Image-Pro Plus software.The density of NeuN positive RGCs per square millimeter was averaged andcompared in experimental retinas treated with 1 μM AlloP or 1 μMent-AlloP at 75 mmHg and control retinas incubated with aCSF at 75 mmHgby Dunnett's multiple comparison test. RGC counts were analyzed usingImage-Pro Plus software. NeuN positive RGCs were determined as themeasurement of 5 different areas from three eyes per experimentalcondition.

Apoptosis—To visualize apoptotic cells, a DeadEnd™ Colorimetric TUNEL(TdT-mediated dUTP Nick-End Labeling) System (Promega, Madison, WI) wasused according to the manufacturer's instructions. The nuclei werecounterstained with DAPI. After the length of each retinal section wasmeasured (Image-Pro Plus software), the cells were counted in the wholesection length and the number of cells was normalized per 200 μm ofretinal section. The number of apoptotic cells was evaluated byDunnett's multiple comparison test to determine changes in the densityof apoptotic cells among experimental retinas treated with 1 μM AlloP or1 μM ent-AlloP at 75 mmHg and control retinas incubated with aCSF at 75mmHg. Apoptotic cells s were determined as the measurement of 5different areas from three eyes per experimental condition.

Western Blot Analysis—At the end of each experiment, the posteriorsegments of the eye were placed on a flat cutting surface and immersedin ice-cold aCSF. With a surgical blade, the retina was carefully andgently detached from the sclera with a fine forceps. The isolatedretinas were frozen at −80° C. Retinas were then homogenized in lysisbuffer solution (CelLytic MT; Sigma-Aldrich, Inc.) with proteaseinhibitor cocktail (Sigma-Aldrich, Inc.), prepared according to themanufacturer's instructions. The tissue extracts were ultrasonicated andclarified by centrifugation at 12,000 g for 20 minutes at 4° C. Theprotein concentrations in supernatants were assayed (Quant-iT assay kit;Invitrogen Corp., Carlsbad, CA). Twenty micrograms of retinal extractwere subjected to SDS polyacrylamide gel electrophoretic analysis. Theproteins were then transferred to a PVDF. Immunoblots by polyclonalrabbit anti-LC3B antibody (MBL Life Science) or anti-SQSTMl/p62 mousemonoclonal antibody (Abcam) were visualized using WesternBreezeChemiluminescent Immunodetection system (Invitrogen) with the exposuretime to autoradiograph film (MXJB Plus; Kodak, Rochester, NY) adjustedto avoid over- or undersaturation. Precision Plus Protein™ WesternStandards (Bio-Rad, Hercules, CA) followed by Precision Protein™ strepTactin-AP conjugate (Bio-Rad) was used for a molecular marker of LC3.MagicMark™ XP Western Protein Standard (Invitrogen, Carlsbad, CA) wasused for a molecular marker of p62. Immunoblot data are normalized toβ-actin levels in the same sample.

The density of Western blot bands was quantified using Image-Pro Plussoftware. Quantification was adjusted to protein expression at 10 mmHgafter normalization to the β-actin band at each pressure, and therelative gray-scale value was calculated by densitometric analysis ofthe obtained bands. At least four independent experiments were performedfor each condition, and the results are presented as relative unitsnumerically. Differences in expression levels were evaluated usingWilcoxon Rank-Sum Test, Tukey's or Dunnett's multiple comparison test.Sample numbers were four to five per each experimental condition.

Chemicals—AlloP was purchased from Wako Pure Chemical Industries, Ltd.(Cat #596-30841, CAS. NO 516-54-1; Osaka, Japan), and ent-AlloP wassynthesized by KK and DFC. 3-MA was purchased from Wako Pure ChemicalIndustries, Ltd. (CAS. No 5142-23-4, Cat #518-92041). All otherchemicals were purchased from Sigma-Aldrich Corp. or Nacalai Tesque(Kyoto, Japan). AlloP and ent-AlloP were dissolved in dimethyl sulfoxide(DMSO) as a 10 mM stock solution.

Statistics—Data were double-checked and analyzed using a biomedicalstatistical computer program(http://www.gen-info.osaka-u.ac.jp/MEPHAS/dunnett.html) on a personalcomputer. Descriptive statistical results were presented using the meanvalues (mean)±standard deviation (SD). For comparison with controls thatwere incubated in drug free aCSF at 10 mmHg, Dunnett's multiplecomparison test was used, depending on sample numbers. For comparisonwith both the control and other conditions, Tukey's multiple comparisontest was used. For all analyses, p values were considered statisticallysignificant, when the values were less than 0.05 (two-tailed).

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters are be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of some embodiments of the presentdisclosure are approximations, the numerical values set forth in thespecific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) areconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or to refer to the alternativesthat are mutually exclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and may also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand may cover other unlisted features.

All methods described herein are performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (e.g. “such as”)provided with respect to certain embodiments herein is intended merelyto better illuminate the present disclosure and does not pose alimitation on the scope of the present disclosure otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member is referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group are included in, or deleted from,a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

To facilitate the understanding of the embodiments described herein, anumber of terms are defined below. The terms defined herein havemeanings as commonly understood by a person of ordinary skill in theareas relevant to the present disclosure. Terms such as “a,” “an,” and“the” are not intended to refer to only a singular entity, but ratherinclude the general class of which a specific example may be used forillustration. The terminology herein is used to describe specificembodiments of the disclosure, but their usage does not delimit thedisclosure, except as outlined in the claims.

All of the compositions and/or methods disclosed and claimed herein maybe made and/or executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of the embodiments includedherein, it will be apparent to those of ordinary skill in the art thatvariations may be applied to the compositions and/or methods and in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit, and scope of the disclosure. Allsuch similar substitutes and modifications apparent to those skilled inthe art are deemed to be within the spirit, scope, and concept of thedisclosure as defined by the appended claims.

This written description uses examples to disclose the disclosure,including the best mode, and also to enable any person skilled in theart to practice the disclosure, including making and using any devicesor systems and performing any incorporated methods. The patentable scopeof the disclosure is defined by the claims, and may include otherexamples that occur to those skilled in the art. Such other examples areintended to be within the scope of the claims if they have structuralelements that do not differ from the literal language of the claims, orif they include equivalent structural elements with insubstantialdifferences from the literal language of the claims.

1. A composition for prevention or treatment of a neurodegenerativecondition, the composition comprising a neurosteroid, a syntheticenantiomer of a neurosteroid, or a combination thereof.
 2. Thecomposition of claim 1, further comprising a saline solution.
 3. Thecomposition of claim 1, further comprising(2-Hydroxypropyl)-β-cyclodextrin.
 4. The composition of claim 1, whereinthe neurosteroid is allopregnanolone and the synthetic enantiomer of theneurosteroid is ent-allopregnanolone.
 5. The composition of claim 1,wherein the neurosteroid, the synthetic enantiomer of the neurosteroid,or the combination thereof has a concentration of from about 10 nM toabout 100 μM.
 6. The composition of claim 1, wherein the compositioncomprises both the neurosteroid and the synthetic enantiomer of theneurosteroid. 7-20. (canceled)
 21. The composition of claim 1, whereinthe neurosteroid is selected from the group consisting of cholesterol,pregnenolone, progesterone, pregn-5α-ane-3,17-dione,pregn-5β-ane-3,17-dione, androsterone, etiocholanolone, andtetrahydrodeoxycorticosterone.
 22. The composition of claim 1, whereinthe synthetic enantiomer of the neurosteroid is selected from the groupconsisting of ent-cholesterol, ent-pregnenolone, ent-progesterone,ent-pregn-5α-ane-3,17-dione, ent-pregn-5β-ane-3,17-dione,ent-androsterone, ent-etiocholanolone, andent-tetrahydrodeoxycorticosterone.
 23. The composition of claim 1,wherein the neurosteroid is progesterone and the synthetic enantiomer ofthe neurosteroid is ent-progesterone.
 24. The composition of claim 6,wherein the combination thereof has a concentration of from about 10 nMto about 100 μM.
 25. The composition of claim 1, wherein the compositioncomprises the synthetic enantiomer of the neurosteroid or thecombination of the neurosteroid and the synthetic enantiomer of theneurosteroid.